Effects of Sr²⁺ and botulinum toxin on stimulation-induced changes in NADH and Ψ_m The stimulation-induced decrease in NADH autofluorescence and partial depolarization of Ψ_m persist when bath Ca²⁺ is replaced by 2 mm Sr²⁺ (A and B), and when transmitter release is blocked by 40 nm BoTxA (C and D). Records in A and C were recorded sequentially from the same terminal, and records in B and D from another terminal. n = 3 experiments for A–Cn = 1 experiment for D.

The stimulation-induced decrease in NADH autofluorescence depends on Ca²⁺ influx into motor terminals The response to 50 Hz stimulation disappeared following addition of 2 mm BAPTA to reduce bath [Ca²⁺] (A), or 3 μmω-conotoxin GVIA (ω-CTX) to block N-type Ca²⁺ channels (B). Control and ω-CTX records in B are averages of four trials; ω-CTX records were obtained after 50–105 min of drug exposure. A and B were recorded from separate terminals; n = 2 experiments for both A and B.

Hypothesized mechanism to explain experimental observations during repetitive stimulation under normal conditions (left box) and following amytal-induced partial inhibition of electron transport chain (ETC) activity (right box) Sequential processes above the boxes are common to both conditions; divergence occurs as a result of differing extents of acceleration of ETC activity in normal and inhibited mitochondria in response to the Ψ_m depolarization produced by Ca²⁺ influx. This proposed mechanism helps to explain why repetitive nerve stimulation produced greater Ψ_m depolarization and greater elevation of cytosolic [Ca²⁺] in the presence of amytal.

Effects of oligomycin and amytal on stimulation-induced changes in NADH, Ψ_m and cytosolic (Ca²⁺) Partial inhibition of complex I activity with 5 mm amytal (•) changes motor terminal responses to stimulation by inhibiting the decrease in NADH fluorescence (A and B), increasing Ψ_m depolarization (C) and increasing the elevation of cytosolic [Ca²⁺] (D). Amytal solutions also contained 10 μg ml⁻¹ oligomycin in A, C and D. A, brief exposure to oligomycin alone (⋄) increased the stimulation-induced decrease in NADH fluorescence; this effect was observed in 2 out of 3 experiments. The NADH response to stimulation gradually disappeared during the second hour of oligomycin exposure (not shown). B, the amytal-induced inhibition of the NADH response to stimulation is reversible. C, amytal increases Ψ_m depolarization during stimulation (averages from four to five trains recorded in two terminals in the same preparation). D, amytal increases the stimulation-induced elevation of cytosolic [Ca²⁺] in a terminal in which the cytosol was filled with fluo-4 dextran. Amytal produced a slight increase in prestimulation net fluorescence in this terminal (data not shown). In A–D drugs were present for ≥ 20 min before the illustrated records were taken.

Repetitive stimulation produces a partial, reversible depolarization of Ψ_m that depends on Ca²⁺ influx into motor terminals Ψ_m depolarization was indicated by an increase in Rh123 fluorescence (due to unquenching). A, Rh123 fluorescence changes in response to a 50 Hz, 40 s stimulus train (left), followed by addition of 1 mm cyanide (CN, right). A second 50 Hz stimulus train applied following CN exposure produced no further increase in fluorescence. The CN-induced fluorescence increase probably underestimated the CN-induced depolarization of Ψ_m, because of loss of Rh123 from the terminal via diffusion into the axon and bath (Nicholls & Ward, 2000). B and C, the stimulation-induced increase in Rh123 fluorescence was inhibited by adding 2 mm BAPTA (B) or 3 μmω-conotoxin GVIA (ω-CTX) to the physiological saline (C). The illustrated stimulation-induced partial Ψ_m depolarization was not detected in a previous study (David, 1999) that used a less-sensitive imaging system. A–C were recorded from different terminals; n = 1 experiment for A; n = 2 experiments for B and C.

The stimulation-induced depolarization of Ψ_m and decrease in NADH fluorescence return to baseline faster than the increase in matrix [Ca²] Changes in matrix [Ca²⁺] were measured using Xrhod-5F loaded into the mitochondrial matrix as described in the Methods. A, changes in mitochondrial [Ca²⁺] (upper trace) and Ψ_m (lower trace) recorded simultaneously from the same motor terminal (n = 2 experiments). B, sequentially recorded changes in NADH fluorescence (circles, lower traces) and matrix [Ca²⁺] (diamonds, upper traces) recorded from another terminal. Responses to a single 20 s train (open symbols) and two sequential 20 s trains (filled symbols) are superimposed. C, changes in Rh123 fluorescence recorded from a different terminal following a two-train stimulation pattern as in B. In A the stimulation-induced increase in Rh123 fluorescence was greater than that illustrated other figures. A likely explanation for this is that in this terminal the regions of interest in which Rh123 fluorescence was analysed were defined as those regions that showed a stimulation-induced increase in Xrhod-5F fluorescence. This procedure improved the spatial resolution and consequently the signal-to-noise ratio of the Rh123 signal by reducing the amount of Rh123 fluorescence recorded from non-responsive regions. In B the prolonged poststimulation plateau of Xrhod-5F fluorescence was not due to saturation of the indicator, but rather to the powerful matrix buffering system described in the text. In B the NADH records were collected first, then the mitochondrial matrix was loaded with Xrhod-5F; the small, rapid initial decay in Xrhod-5F fluorescence following each stimulus train may have arisen from a small amount of Xrhod-5F remaining in the cytosol.

Stimulation of lizard motor terminals evokes a reversible decrease in NADH autofluorescence A, decrease in NADH fluorescence and increase in FAD⁺ fluorescence (change in ratio of fluorescence to the averaged resting fluorescence prior to stimulation; ΔF/F₀) induced by 100 s of 50 Hz stimulation in a terminal. Vertical dashed lines mark the beginning and end of the stimulus train, which was longer than the trains used in subsequent figures. The sampling rate was 2.3 min⁻¹. B and C, reciprocal changes in net fluorescence (arbitrary units) of NADH and FAD⁺ in a terminal exposed first to 2 μm CCCP to induce maximal oxidation and then to 10 mm cyanide to induce maximal reduction of mitochondrial pyridine nucleotides. D and E, two patterns of NADH fluorescence (F/F₀) change evoked by 50 s of 50 Hz stimulation. In the terminal in D, NADH fluorescence simply returned to baseline following stimulation, whereas in the terminal in E there was a small, prolonged poststimulation increase in NADH fluorescence. F, changes in net NADH fluorescence in a terminal that was (in succession) stimulated at 50 Hz, exposed to Na cyanide to maximize NADH, returned to normal medium, and exposed to CCCP to minimize NADH fluorescence (n = 3 experiments). Image collection was discontinued during solution changes; points between 500 and 1250 s are not shown. Data in A–C were collected with an imaging system that included a Leica DM IRBE fluorescence microscope, a TILL Photonics monochromator, and a Hamamatsu ER CCD camera. Data in D–F and all subsequent figures were obtained using the imaging system described in the Methods.

Partial inhibition of ETC activity has a greater effect on mitochondrial function in stimulated than in resting terminals Simplified equivalent circuit of the mitochondrial inner membrane (A) allows prediction of how the number of H⁺-extruding complexes affects Ψ_m depolarization (B) and ETC H⁺ extrusion (C) in the absence (rest) or presence (stimulated) of Ca²⁺ influx into motor terminal mitochondria. Partial inhibition of ETC activity (e.g. due to inhibiting complex I) reduces the stimulation-induced increase in H⁺ extrusion and increases the stimulation-induced depolarization of Ψ_m. In A, H⁺ extrusion by respiratory complexes I, III and IV is modelled as current through R_ETC driven by the battery electromotive force (Emf), where R_ETC is the resistance of all proton-extruding complexes arranged in parallel. H⁺ current into the matrix via H⁺ leak and the F₁,F₀-ATPase (complex V) occurs via membrane resistance (R_m). Nerve stimulation is simulated by closing the switch in the Ca²⁺ branch of A, allowing Ca²⁺ to flow into the mitochondrial matrix via the uniporter (R_Ca). In B and C, a reduction in the number of active complexes is simulated by increasing R_ETC. The length of the vertical arrows in B and C indicates the magnitude of the changes in Ψ_m and I_ETC (respectively; I_ETC is current through ETC complexes) associated with Ca²⁺ influx into inhibited (filled arrows) and non-inhibited (open arrows) mitochondria. B and C were calculated by applying Kirchoff's and Ohm's laws to this circuit [for B, ΔΨ_m= Emf/(1 +R_ETC(R_m⁻¹+R_Ca⁻¹); for C, I_ETC= (Emf –ΔΨ_m)/(R_ETC)]. Parameters used in the simulations of B and C (adjusted for 1 mg protein) were Emf = 200 mV, R_m= 3 × 10³Ω, R_Ca= 7 × 10²Ω (value at half-maximal conductance of the uniporter, Magnus & Keizer, 1997). R_ETC in normally respiring mitochondria is ∼10 Ω (Nicholls, 1974). Capacitance (0.14 MF) was omitted because the time constant of this circuit is very short compared to the duration of Ca²⁺ influx. This simplified model ignores the chemical components of the driving forces for Ca²⁺ and H⁺ fluxes, assumes no changes in the concentrations of substrate or these ions, and ignores possible changes in Emf (e.g. due to depletion of NADH) and possible saturation of respiratory chain complexes. A similar, albeit more complex, equivalent circuit has been presented by Lemeshko (2002).